RPMI medium supplemented

Examine the cells under the microscope to ensure that all the fibroblasts are attached to the tissue culture dishes (Fig. la). Wash the layer of fibroblasts twice with 5 ml of pre-warmed PBS. Seed 2x10 CLL cells (ratio 10 1 10 CLL cells per NIH3T3 cell Fig. lb) on WT and CD154 N1H3T3 in 5 ml complete RPMI medium supplemented with 10 ng/ml of lL-4 (only in CD154 NIH3T3-coated tissue culture dishes) and incubate for 24 h in a humidified atmosphere of 5% CO at 37°C (see Notes 7 and 8). 

Interleukin-2 (IL-2). Used as solution of 200 U/mL in RPMI medium supplemented with 15% heat-inactivated FCS, 2 mM L-glutamine, 0.1% sodium bicarbonate, and 20 pg/ml. gentamycin. This solution is stored at -20°C. 

Fig. 4. Wortmannin-induced apoptosis in LNCaP cells. (A) Dose- and time-dependent effect of wortmannin on cell viability of LNCaP cells in RPMI medium supplemented with 0.1% fetal bovine serum. The data represent the mean of three independent determinations. (B) Dose- and time-dependent effect of wortmannin on DNA fragmentation. The first lane is a 100-bp DNA ladder. (C) Wortmannin (0.5 pM) inhibits Akt phosphorylation in a time-dependent manner in LNCaP cells. In contrast, wortmannin was not effective against PC-3 cells. Source Reprinted from Reference 74 with permission from Elsevier.

To prepare the differentiated HL-60 cells 2x10, HL-60 cells are inoculated into 10 ml antibiotic-free RPMI medium supplemented with 10% FBS and 1.3% endotoxin-low DMSO is added. Cells are incubated at 37 ° C, 5% CO2 for 5—7 days. The morphology of cells will change during the process of differentiation from a perfecdy round shape to a randomly irregular shape with multiple protrusions on the cell membrane. 

CLL Cells isolated from peripheral blood by density gradient centrifugation were cultured for 12 hr in the absence or presence of 1 iM CdA in nicotinamide-free RPMI medium, supplemented with L-glutamine 2 mM and 20% autologous plasma. Neutralized perchloric acid extracts were analyzed for NAD content by an enzyme cycling assay, and for ATP content by HPLC. 

Tissue Culture Assay. Kogure et al. (48) report a novel tissue culture assay for detecting several types of sodium channel blockers. The mouse neuroblastoma cell line ATCC CCL 131 is grown in RPMI 1640 supplemented with 13.5% fetal bovine serum and 100 pg/ml gentamycin, in an atmosphere of 5% C0 95% air at 37 C. Ninety-six well plates are seeded with 1 x 10 cells in 200 pi of medium containing 1 mM ouabain and 0.075 mM veratridine. Veratridine and ouabain cause neuroblastoma cells to round-up and die. In the presence of sodium channel blockers (e.g., TTXs or STXs), the lethal action of veratridine is obviated and cells retain normal morphology and viability. An important feature of this assay is that a positive test for sodium channel blockers results in normal cell viability. Since bacterial extracts can contain cytotoxic components, this assay offers an advantage over tests that use cell death as an endpoint. The minimum detectable level of TTX is approximately 3 nM, or approximately 1/1000 mouse unit. 

Primary CLL cells purified from peripheral blood of volunteer patients (see Note 3) CLL cells are purified by Ficoll gradient centrifugation, by negative selection, or by a combination of both (see Note 4). CLL cells can be used immediately or frozen in 90% heat/inactivated FBS supplemented with 10% DMSO and stored at -80°C or in liquid nitrogen for short-and long-term storage, respectively. CLL cells are cultured in complete RPMI medium and maintained in a humidified atmosphere of 5% CO at 37°C (see Note 5). 

Quickly thaw a frozen aliquot of NS-O cells either in a 37°C water bath or between the palms of the hands. Once the pellet has melted, add 1 mL of 37°C RPMI1640 medium supplemented with 10% FBS and draw up into a Pasteur pipet. Transfer the cells to a 225-cm T flask and add 50 mL of RPMI 1640 medium containing 5% FBS and place in a tissue culture incubator 37°C/5% C02. 

CCRF-CEM T-lymphoblastoid cells (ATCC CCL 119) were cultivated in RPMI 1640 medium supplemented with L-glutamine (0.3 g/L) containing 10% bovine serum using 24-well tissue culture plates. The cells were seeded at 10s mL 1 and after a 24-h incubation period (C02 atmosphere, 37 °C) tested compounds were added at five different concentrations. The endpoint of the cell growth was 72 h following the drug addition. An appropriate aliquot from every dish was then counted (cell counter Serono 150+). The inhibitory potency of the compounds tested was expressed as IC50 values. 

RPMI medium 1640 (Gibco, Invitrogen, Carlsbad, CA) supplemented with 25% (v/v) of human serum (Fisher BioReagents, Fisher Scientific, Pittsburg, PA) (see Note 2). 

The mixed lymphocyte reaction (MLR) test was performed in 96-well flat-bottomed microtiter plates. Selected experimental agents were prepared as 10 mM stock solution in DMSO and a 50-fold dilution of this was prepared in RPMI. Serial dilutions were prepared from this subsequent solution and 10 xl of the diluted stock was added to the well to give concentrations in the assay starting at 9.5 xm. In each well was placed 1.5 x 105 donor cells to give a final volume of 0.2 ml RPMI 1640 medium supplemented with 10% human serum, 2mM L-glutamine, and penicillin/streptomycin. Cells were incubated at 37°C in a humidified atmosphere containing 5% carbon dioxide for 120 hours. 3H-Thymidine (0.5 xCi) was added in the final 6 hours of incubation and cell radioactivity levels determined, which were indicative of T-cell proliferation. 

An IR spectrum of freshly prepared RPMI 1630 cell culture medium supplemented with 5% calf serum, the medium used routinely to culture viable cells, is presented in Figure 1. Figure 1 is an IR spectrum of centrifugally concentrated and washed EAT cells which were cultured in this medium. The IR spectrum of the salt-free residue from the supernatant liquid obtained after centrifuging the cells constitutes Figure... 

Roswell Park Memorial Institute-1640 (RPMI-1640) medium supplemented with 10% fetal calf serum (PCS)... 

A549 cells were grown in RPMI-1640 medium supplemented with 10% FBS in a humidified 5% CO atmosphere. 

I-labelled siRNA solution 4.8 pig/ml total siRNA (GFP-22 siRNA, Qiagen) comprising 2 x 10 CPM/ml I-labelled siRNA from Step 3.3 in RPMI medium without supplements, or whatever solvent of interest. 

Adherent EJ urinary bladder carcinoma cells (see Note 7) are cultured routinely in RPMI-1640 medium supplemented with 10% FBS and L-glutamine and reset by hot trypsiniza-tion two or three times per week. For the experiments, only recently established cultures can be used advisably before the seventh passage. 

For the competition assays, AR42J cells were seeded at a density about 800 000 cells per well on six-well plates and incubated for 24-48 h. Cells were then washed twice with RPMI-1640 medium supplemented with 10% FCS and antibiotics. The cells were supplied with fresh medium, and the radiolabelled preparation, corresponding roughly to 200 fmol peptide in 150 gL PBS/0.5% BSA buffer, was added. The competitor (Sandostatin) was added in ten concentrations (0.01, 0.1, 1.0, 3.0,10, 30,100, 300,1 000,10 OOOnM) in a total volume of 150 gL of PBS/0.5% BSA buffer. The cells were incubated for 2 h at... 

The complete medium to maintain the Jurkat T cell line is RPMI 1640 supplemented with 10 % FBS, 2 mM glutamine and lx penicillin-streptomycin. 

Molecular Weight Cutoff (RPMI-1640 Supplemented Serum-Free Medium)... 

In Vitro Cell Culture. Mouse leukemia L1210 cells (designated K25) were grown in RPMI 1630 medium supplemented with 20% heat-inactivated fetal calf serum. V79 Chinese hamster lung cells were grown in o-MEM medium with 5% fetal calf serum In 7.5% CO2. The human cell lines were grown in Eagle s minimal essential medium with 10% fetal calf serum. 

Cell Culture. The HBV-producing human blastema cell line (HepG2.2.15) (253) was maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (PCS), 2 mM glutamine, 100 units/ml penicillin, 100 /tg/ml streptomycin and 350 ng/tnl gentamicin sulfate (G418) at 37°C under 5% CO2 in air. Similarly, HepG2 cells were maintained in RPMI 1640 medium supplemented with 10% PCS, 2 mM glutamine, 100 units/ml penicillin and 100 tg/ml streptomycin. 

A chronically HIV-1lav-i infected T-cell line (CEM/LAV-1) was maintained at 37 °C in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS) containing 100 lU/ml penicillin and 100 pg/ml streptomycin in 5% C02. 

HL60 cell culture medium. RPMI 1640 supplemented with 25 mM HEPES, 10% FBS, 100 lU/ml penicillin, 100 pg/ml streptomycin G, 50 pg/ml gentamycin. 

RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (50 U/ml), and streptomycin (50 mg/ml) (Invitrogen, Carlsbad, CA). 

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